Abstract: The surface relaxation, electronic properties, and copper activation on sphalerite (110) surface were studied by using density functional theory (DFT), in order to investigate the effect of iron content on the iron-bearing sphalerite. The results showed that for the sphalerite with low iron content, the relaxation of zinc atoms and iron atoms on the sphalerite (110) surface to the interior of the surface becomes smaller, which reduces the steric hindrance; the electronic density of states and the energy band structure showed that the electronic activity of iron is relatively strong and there is no spin polarization, which means that low iron content may favor the flotation of sphalerite. For sphalerite with high iron content, the iron atoms on the surface relax more towards the interior, which increases the steric hindrance; the electronic state density and energy band structure showed that the activity of iron is not high, and spin polarization occurs, which is not conducive to flotation. However, iron on the sphalerite (110) surface is not easily replaced by copper, and the more iron content, the less conducive to the replacement of copper. This study explains the effect of iron content affecting surface properties and copper activation on sphalerite (110) surface at an atomic level, which provides theoretical guidance for the flotation of iron-bearing sphalerite.